Báo cáo khoa học: Investigating the role of the invariant carboxylate residues E552 and E1197 in the catalytic activity of Abcb1a (mouse Mdr3) docx

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Investigating the role of the invariant carboxylate residuesE552 and E1197 in the catalytic activity of Abcb1a(mouse Mdr3)Isabelle Carrier and Philippe GrosDepartment of Biochemistry and McGill Cancer Centre, McGill University, Montreal, CanadaMultidrug resistance (MDR) is of major concern in thetreatment of many important human diseases such ascancer, schizophrenia and infections by micro-organ-isms, including HIV [1–3]. MDR is characterized bycross-resistance to structurally and functionally unre-lated chemicals. Overexpression of membrane trans-porters of wide substrate specificity is the mostcommon cause of MDR. These transporters includemembers of the ATP-binding cassette (ABC) proteinsuperfamily, such as P-glycoprotein (Pgp, ABCB1),multidrug resistance-associated protein (MRP, ABCC1)and breast cancer resistance protein (BCRP, ABCG2)[4]. With 48 members in humans, 56 in the fly (Droso-phila melanogaster), 129 in plants and well over 300 inbacteria, the ABC transporter superfamily is one ofthe largest and most conserved gene families known[5,6]. Mutations in about half of the 48 humanmembers cause diseases and phenotypes includingMDR, and make this family of proteins of great clini-cal interest [7]. Diseases include Tangier disease(ABCA1), cystic fibrosis (ABCC7) and sitosterolemia(ABCG5, ABCG8), to name a few.KeywordsABC transporter; Abcb1a; ATP hydrolysis;catalytic mechanism; nucleotide-bindingdomainCorrespondenceP. Gros, Department of Biochemistry andMcGill Cancer Centre, McGill University,McIntyre Medical Sciences Building, Room907, 3655 Sir William Osler Drive, Montre´al,Que´bec H3G 1Y 6, CanadaFax: +1 514 398 2603Tel: +1 514 398 7291E-mail: philippe.gros@mcgill.ca(Received 6 February 2008, revised 26March 2008, accepted 24 April 2008)doi:10.1111/j.1742-4658.2008.06479.xThe invariant carboxylate residue which follows the Walker B motif(hyd4DE ⁄ D) in the nucleotide-binding domains (NBDs) of ATP-bindingcassette transporters is thought to be involved in the hydrolysis of thec-phosphate of MgATP, either by activating the attacking water moleculeor by promoting substrate-assisted catalysis. In Abcb1a, this invariant car-boxylate residue corresponds to E552 in NBD1 and E1197 in NBD2. Tofurther characterize the role of these residues in catalysis, we created inAbcb1a the single-site mutants E552D, N and A in NBD1, and E1197D,N and A in NBD2, as well as the double-mutant E552Q ⁄ E1197Q. In addi-tion, we created mutants in which the Walker A K fi R mutation knownto abolish ATPase activity was introduced in the non-mutant NBD ofE552Q and E1197Q. ATPase activity, binding affinity and trapping proper-ties were tested for each Abcb1a variant. The results suggest that the lengthof the invariant carboxylate residue is important for the catalytic activity,whereas the charge of the side chain is critical for full turnover to occur.Moreover, in the double-mutants where the K fi R mutation is intro-duced in the ‘wild-type’ NBD of the E fi Q mutants, single-site turnoveris observed, especially when NBD2 can undergo c-Picleavage. The resultsfurther support the idea that the NBDs are not symmetric and suggest thatthe invariant carboxylates are involved both in NBD–NBD communicationand transition-state formation through orientation of the linchpin residue.AbbreviationsABC, ATP-binding cassette; Abcb1a, mouse P-glycoprotein ⁄ Mdr3 ⁄ Mdr1a; IC, invariant carboxylate; MDR, multidrug resistance; NBD,nucleotide-binding domain; NBD1, N-terminal nucleotide-binding site; NBD2, C-terminal nucleotide-binding site; Pgp, P-glycoprotein; TMD,transmembrane domain; Vi, ortho-vanadate (VO4)).3312 FEBS Journal 275 (2008) 3312–3324 ª 2008 The Authors Journal compilation ª 2008 FEBSThe structural subunit which defines ABC transport-ers is composed of one transmembrane domain(TMD), formed by six putative transmembrane a heli-ces and one cytosolic nucleotide-binding domain(NBD) [8,9]. Usually, a complete ABC transporter isrepresented by various combinations of four domains,of which two are TMDs and two are NBDs [10]. Thefour domains of this membrane-associated complexcan be assembled from two to four separate proteinsubunits (most prokaryotes) or arranged in one singlepolypeptide (most eukaryotes). Crystallization of theABC transporters Sav1866 and ModBC, in the absenceand presence of nucleotide, has provided good struc-tural models for ABC transporters in the lipid bilayerand the changes associated with dimerization andopening of the NBDs [11–14]. In these 3D structures,it is thus possible to observe the position of eacha helix in the TMD and establish which helices inter-act. Also, in the structures where nucleotide is present,dimerization of the NBDs is demonstrated, asobserved for other NBDs that were purified withouttheir TMDs [15–19]. In ABC transporters, the TMDsform the translocation pathway and the NBDs hydro-lyze ATP to energize transport. Based on the fact thatATP hydrolysis by ABC transporters is highly coopera-tive, it has been suggested that the two NBDs functionas a dimer in the translocation process [20,21]; this hasnow been firmly established by several crystal struc-tures [11,15,17].Whereas the TMDs are responsible for allocritetransport, it is the energy from ATP binding andhydrolysis, by the NBDs, that drives this transport.A high degree of sequence and structural conser-vation is observed for NBDs across the family. TheNBD is an L-shaped protein with a two-domainarchitecture: the first is the catalytic domain, com-posed of an ABC (ABCb) and a RecA-like sub-domain, and contains the nucleotide-binding site; thesecond is the helical domain (ABCa), which interactswith the TMD and is unique to ABC transportersbecause of an insertion of  70 residues between thetwo Walker motifs [22,23]. Each NBD contains sev-eral conserved sequence motifs: the Walker A andB motifs, the signature or LSGGQ motif and theA-, D-, H- and Q-loops. These motifs are positionedaround the bound nucleotide and help to positionand maintain it in the active site. In particular, theWalker A motif wraps around the b-phosphate ofbound nucleotide [22], the Walker B motif is respon-sible for coordinating the essential Mg2+cofactor[22,24,25], the signature sequence contacts thec-phosphate of the bound nucleotide across thedimer [15] and the aromatic residue of the A-loopstacks against the adenine moiety of boundnucleotide and provides further stabilization andspecificity [26,27]. The D-loop is thought to beinvolved in NBD–NBD communication [28,29]. TheH-loop has recently been hypothesized to be directlyinvolved in hydrolysis of the c-phosphate by posi-tioning the terminal phosphate in the correct orienta-tion for attack by the catalytic water molecule [30].And finally, the Q-loop, whose glutamine residueinteracts with the putative catalytic water and a helixextending from the TMD, may be involved in signaltransduction between the TMD and NBD, by sens-ing either hydrolysis of the terminal phosphate orthe presence of substrate in the drug-binding site[31,32].Although recent successes in solving the crystal struc-tures of ABC transporters have laid the foundation fora new era of studies using structure-guided mutagenesis,many issues relating to the mechanism of action of ABCtransporters remain obscure. An important issue is thecatalytic mechanism of ATP hydrolysis by the twoNBDs, which can be further subdivided into twomajor components. The first pertains to the actual cleav-age of the terminal phosphate and the second toNBD–NBD communication. Two models of catalysisby ABC transporters are currently accepted: (a) generalbase [23,33,34] and (b) substrate-assisted [28,30].Interestingly, both models involve the invariantcarboxylate (IC) residue which immediately follows theWalker B aspartate, although it performs different tasksin each case. In the former model, the IC is the catalyticresidue which coordinates and activates the attackingwater molecule that cleaves the terminal phosphate ofbound ATP. In the latter model, the IC is part of acatalytic dyad, along with the histidine residue of theH-loop, and positions the ‘linchpin’ histidine in thecorrect orientation such that all atoms are then inposition to favor abstraction of a proton from theattacking water molecule by the bound ATP, whichresults in cleavage of the terminal phosphate by theaforementioned water molecule. At present, it istempting to favor substrate-assisted catalysis as themechanism of action of ABC transporters becausemutating the IC(s) in different enzymes is incompatiblewith a role for this residue in general base catalysis[35–38].In order to investigate further the role of the IC inthe catalysis of ABC transporters, we created, inAbcb1a, six single-site mutants (E552D, N and A, andE1197D, N and A,) and three double-mutants(E552Q ⁄ E1197Q, E552Q ⁄ K1072R, K429R ⁄ E1197Q).The ATPase activity, binding affinity and trappingproperties were tested for each Abcb1a variant.I. Carrier and P. Gros Abcb1a catalytic mechanismFEBS Journal 275 (2008) 3312–3324 ª 2008 The Authors Journal compilation ª 2008 FEBS 3313ResultsIn a previous study, analysis of mutants E552Q andE1197Q of mouse Abcb1a suggested that single-siteturnover did occur in these mutant enzymes and thatADP release was the most likely step impaired by themutations. Interpretation of these results alsosuggested that the two NBDs of Pgp were notfunctionally equivalent [39]. Studies by other groupsalso showed that these IC residues are not directlyinvolved in the hydrolysis of the terminal phosphate ofATP and it was determined that the ICs either playeda role in NBD–NBD communication [36] and ⁄ ornormal transition state formation following NBDdimerization [38,40]. In this study, we investigatedfurther the role of these two IC residues in thecatalytic mechanism of Abcb1a. For this, wild-typeand the Abcb1a mutants E552D, E552N, E552A,E1197D, E1197N, E1197A, E552Q ⁄ E1197Q (Q ⁄ Q),E552Q ⁄ K1072R (Q ⁄ R) and K429R ⁄ E1197Q (R ⁄ Q),were expressed in the yeast Pichia pastoris as recombi-nant proteins bearing an inframe polyhistidine tail(His6) at the C-terminus. Protein purification fromlarge-scale methanol-induced liquid cultures of P. pas-toris was performed by detergent extraction fromenriched membrane fractions, followed by affinity andanion-exchange chromatography on Ni2+-NTA andDE52-cellulose resins, respectively [41]. Using this pro-tocol, all proteins could be purified in large amounts(0.4–1.7 mg per 6 L culture) in a stable form and at ahigh degree of purity (>95%; Fig. 1).Steady-state ATP hydrolysis by the purified proteinsactivated with Escherichia coli lipids and dithiothreitolwas determined by measuring Pirelease [42], in theabsence or presence of MDR drugs or Pgp inhibitorsthat are known to stimulate the ATPase activity ofPgp. Wild-type Abcb1a has low basal ATPase activity(0.13 lmolÆmin)1Æmg)1), which can be stronglystimulated (12- to 18-fold) by verapamil andvalinomycin (to 2.38 and 1.66 lmolÆmin)1Æmg)1) [39].The nine Abcb1a mutants all showed very low ATPaseactivity with values comparable to those obtained inan assay in which all reagents were added except forthe protein. In addition, this low basal activity was notstimulated by the addition of drug substrates (data notshown). Thus, all mutants appear to have no steady-state ATPase activity, although we cannot exclude thepossibility that they retain very low levels of suchATPase activity, as seen in Tombline et al. [38].However, such levels would be below the threshold ofaccurate detection and reproducibility of our currentassay; and would represent < 1% of the activity ofthe wild-type enzyme.We then determined, by photoaffinity labeling,whether any of the mutations altered the apparentbinding affinity of Abcb1a for ATP. Purified and acti-vated proteins were incubated with increasing amountsof 8-azido-[a32P]ATP in the presence of Mg2+(10 minon ice), followed by UV irradiation. Unincorporatedligand was removed by centrifugation and labeled pro-teins were resolved by SDS ⁄ PAGE. The gels werestained with Coomassie Brilliant Blue, to quantifyamount of protein loaded (not shown), dried and thensubjected to autoradiography (Fig. 2). Binding and8-azido-[a32P]ATP photo-crosslinking was specific toAbcb1a and increased proportionally with the amountof 8-azido-[a32P]ATP present in the reaction. The [32P]incorporation profile over several experiments wasquantitatively similar for all mutants and was also verysimilar to that seen for the wild-type. These resultssuggest that the introduced mutations do not have aFig. 1. Purification of NBD mutants from P. pastoris membranes.Two micrograms of purified (concentrated DE52 eluate) wild-type-and mutant Abcb1a variants E552A, D, N, E1197A, D, N,E552Q ⁄ E1197Q (Q ⁄ Q), E552Q ⁄ K1072R (Q ⁄ R) and K429R ⁄ E1197Q(R ⁄ Q) were subjected to SDS ⁄ PAGE, followed by staining withCoomassie Brilliant Blue. The position of the molecular massmarkers is given on the left.Fig. 2. Direct photolabeling of purified Abcb1a NBD mutantswith Mg-8-azido-[a32P]ATP. Purified and activated wild-type andmutant Abcb1a variants (E552Q ⁄ E1197Q, E552Q ⁄ K1072R, K429R ⁄E1197Q, E552A, D, N, E1197A, D and N) were UV-irradiated on icein the presence of 3 mM MgCl2and 5, 20 and 80 lM 8-azido-[a32P]ATP. Photolabeled samples were separated on 7.5% SDSpolyacrylamide gels and stained with Coomassie Brilliant Bluefollowed by autoradiography (Experimental procedures). The posi-tion of the molecular mass markers is given on the left.Abcb1a catalytic mechanism I. Carrier and P. Gros3314 FEBS Journal 275 (2008) 3312–3324 ª 2008 The Authors Journal compilation ª 2008 FEBSmajor effect on nucleotide binding to Abcb1a and aretherefore unlikely to cause major non-specific struc-tural changes in the NBDs. This agrees with previousstudies of catalytic residue mutants of the Walker Aand Walker B motifs and of the ICs (K429R, K1072R,D551N, D1196N, E552Q and E1197Q), which severelyaffect the catalytic activity of mouse Abcb1a but havelittle effect on the nucleotide-binding affinity of theprotein [24,39,43]. In addition, this confirms the notionthat residues E552 and E1197 seem to participate inthe catalytic steps after the initial binding of ATP tothe NBDs.Pgp ATPase activity can be stably inhibited by vana-date (Vi), a transition state analogue structurallyrelated to phosphate (Pi) [44]. Trapping of nucleotideby Vi requires both hydrolysis of the bond between theb- and c-phosphates of ATP and release of Pi. Vi canreplace Pionce it is released, capturing ADP in thenucleotide-binding site and forming a long-lived inter-mediate that resembles the normal transition state{MgADPÆVi} [45]. When 8-azido-[a32P]ATP is used asa substrate, this intermediate can be visualized by UVcross-linking [45]. Indeed, Vi-induced trapping of8-azido-[a32P]ADP under hydrolysis conditions (37 °C)has been used as an alternative and highly sensitivemethod to monitor ATPase activity in wild-type andmutant Pgp [24,45]. For wild-type Abcb1a, nucleotidetrapping is completely dependent on the presence ofVi and is strongly stimulated by verapamil and valino-mycin (Fig. 3). Despite the observed lack of ATPaseactivity of the nine mutants analyzed (as measured byPirelease), 8-azido-nucleotide trapping is readily detect-able in these mutants, with the notable exception of theQ ⁄ R double-mutant, which is only very weakly labeled(faint bands seen in the presence of drug upon overex-posure; not shown). For the single-site mutants in bothNBDs, nucleotide trapping either resembles wild-type(E552D and E1197D) or the previously analyzedE552Q (E552N and E552A) and E1197Q (E1197N andE1197A) mutants. In the double-mutants Q ⁄ Q, R ⁄ Qand Q ⁄ R, trapping appears to be drug stimulated butVi independent and occurs most readily in the R ⁄ Qmutant. In fact, the Q ⁄ R enzyme traps nucleotide onlyto a very low extent and visibly only in the presence ofdrugs (± Vi). These results are reminiscent of previousstudies of double-mutants of the IC in human andFig. 3. Photolabeling of Abcb1a NBDmutants by vanadate trapping with Mg-8-azido-[a32P]ATP. Purified and activated wild-type and mutant Abcb1a variants werepre-incubated for 20 min at 37 °C with 5 lM8-azido-[a32P]ATP and 3 mM MgCl2in theabsence or presence of 200 lM vanadate.Verapamil (100 lM) and valinomycin(100 lM) were included as indicated abovethe lanes. Samples were processed forphotolabeling as described in Experimentalprocedures and analyzed by SDS ⁄ PAGE.I. Carrier and P. Gros Abcb1a catalytic mechanismFEBS Journal 275 (2008) 3312–3324 ª 2008 The Authors Journal compilation ª 2008 FEBS 3315mouse enzymes [36,40]. It is interesting to note that thesingle K429R mutant could not trap 8-azido-nucleotideunder any of the conditions tested [24], whereas intro-duction of the E1197Q mutation in its wild-type NBDnow allows for 8-azido-nucleotide to be substantiallytrapped in the protein.We next wanted to determine whether these mutantenzymes were able to hydrolyze the terminal phosphateof bound ATP and form ADP. For this, we used TLCto analyze the nucleotides tightly bound to the proteinfollowing trapping in the presence of Vi and drug, underhydrolyzing (37 °C) and non-hydrolyzing (4 °C) condi-tions. The appearance of a spot corresponding to8-azido-[a32P]ADP was monitored and indicated thathydrolysis did take place. As seen in Fig. 4, 8-azido-[a32P]ADP can be detected following incubation with8-azido-[a32P]ATP and Vi under hydrolysis conditions(37 °C) in all the single-site and double-mutants, withthe exception of the Q ⁄ Q mutant. Production of8-azido-[a32P]ADP in all mutants (except Q ⁄ Q) wastemperature sensitive, as determined by disappearanceof the 8-azido-[a32P]ADP spot when the trappingreaction was carried out at 4 °C, suggesting that the8-azido-[a32P]ADP spot appeared as a result of hydro-lysis of 8-azido-[a32P]ATP. Thus, although the spotcorresponding to 8-azido-[a32P]ADP detected in theQ ⁄ R mutant was faint, it was considered genuine.Because trapping in the double-mutants appears tobe Vi independent, a dose–response assay (0,0.05 lm £ Vi £ 100 lm) was carried out on the R⁄ Qmutant. Figure 5 clearly demonstrates that, unlikeFig. 4. TLC analysis of vanadate-trapped nucleotides in Abcb1aNBD mutants. Purified and activated wild-type and mutant Abcb1avariants were pre-incubated with 5 lM 8-azido-[a32P]ATP and 3 mMMgCl2for 10 min at either 37 or 4 °C in the presence of 200 lMVi and 100 lM verapamil. Unbound ligands were removed byultracentrifugation and washing. The protein pellets were thenresuspended in 8-azido-ATP and precipitated by trichloroacetic acid.Supernatant (0.5 lL) and 125 dpm of standards were applied to aPEI-Cellulose plate following magnesium chelation with EDTA. Theplate was developed in 3.2% (w ⁄ v) NH4HCO3and exposed to film.The asterisk (*) indicates the position of a non-specific radioactivecontaminant present in the commercial preparation of 8-azido-[a32P]ATP.Fig. 5. Photolabeling of Abcb1a NBD mutants with Mg-8-azido-[a32P]ATP and varying concentrations of vanadate. Purified andactivated Abcb1a variants K429R ⁄ E1197Q, E552Q and E1197Qwere pre-incubated with 5 lM 8-azido-[a32P]ATP, 3 mM MgCl2and100 lM VER for 20 min at 37 °C in the absence or presence ofincreasing concentrations of Vi, as indicated above the lanes.Samples were processed for photolabeling as described in Experi-mental procedures and analyzed by SDS ⁄ PAGE. E552Q andE1197Q were included as controls since these mutants displayvarying degrees of Vi-dependence of photolabeling.Abcb1a catalytic mechanism I. Carrier and P. Gros3316 FEBS Journal 275 (2008) 3312–3324 ª 2008 The Authors Journal compilation ª 2008 FEBSE552Q and E1197Q, the R ⁄ Q double-mutant does notrespond to increasing concentrations of Vi.Given that the R ⁄ Q and Q ⁄ Q double-mutants arephotolabeled by 8-azido-[a32P]-nucleotide and thisphotolabeling occurs in a Vi-independent fashion, wewanted to determine in which NBD the 8-azido-nucle-otides were trapped in these proteins. To answer thisquestion, we took advantage of the trypsin-sensitiveregion situated in the linker domain of Abcb1a. Fol-lowing trapping in the absence or presence of Vi andmild-trypsin treatment, the trypsin degradation pro-ducts of the two mutants R ⁄ Q and Q ⁄ Q were resolvedby SDS ⁄ PAGE and immobilized on nitrocellulosemembranes. Immunoblotting of the membranes byPgp-specific antibody C219 reveals that increasing con-centrations of trypsin degrade the enzymes to differentextents and the identity of the fragments could bedetermined by N- and C-terminal-specific antibodies(see Experimental procedures; data not shown). Forthe R ⁄ Q mutant, the two fragments corresponding tothe N- and C-terminal halves of the protein cut at thelinker region could be detected in lanes 2–4 ()Vi and+Vi). Thus, it is possible to observe that the trappednucleotide(s) appears to be exclusively in the MD-7reactive fragment which contains NBD2, both in theabsence and presence of Vi (Fig. 6). For the Q ⁄ Qmutant, the two fragments corresponding to theN- and C-terminal halves of the protein cut at thelinker region could also be detected in lanes 2–4 ()Viand +Vi) and the radiolabel could be detected in eachfragment, both in the absence and presence of Vi(Fig. 6). Because the trapping signal in the Q ⁄ Rmutant was so low, we did not attempt this experimentwith this enzyme.DiscussionDespite the fact that ABC transporters are highly clini-cally relevant and have been studied for well over20 years, many questions about their mechanism ofaction remain partially elucidated. For example, theexact catalytic cycle, the functional symmetry or asym-metry of the NBDs and the types of signals producedthroughout the protein to mediate allocrite transportare still not fully understood. But using the increasingnumber of crystal structures available for ABC trans-porter family members, together with the resultsobtained following mutagenesis of key residues in vari-ous ABC enzymes, a general mechanism of action isbeginning to emerge. One such key residue is theinvariant carboxylate (IC, sometimes called the ‘cata-lytic carboxylate’) that immediately follows the Walk-er B motif in each NBD. This residue was initiallymutated in Abcb1a NBDs and identified a uniquephenotype in which dependence on Vi for trapping of8-azido-nucleotide was partially lost [35]. In mouseAbcb1a, these residues correspond to E552 and E1197in NBD1 and NBD2, respectively. Subsequent studieswith human and mouse enzymes, in which these tworesidues were mutated to other amino acids singly ortogether, or in combination with other mutations, havesuggested that they are not classical catalytic residues,because cleavage of the c-phosphate does occur in theNBD with the mutation at the IC [36,38,39]. More-over, these and other studies suggest that the ICresidues are involved in the formation of the NBDdimer, now recognized as a catalytic intermediate inthe ATP hydrolysis pathway that leads to allocritetransport [38,40,46]. In addition to the E552D,E1197D, E552A, E1197A and E552Q ⁄ E1197Q mutantsalso analyzed in previous studies (mouse and humanFig. 6. Trypsin digestion of Abcb1a NBD mutants photolabeledwith Mg-8-azido-[a32P]ATP in the absence or presence of vana-date. Purified and activated mutant Abcb1a variantsK429R ⁄ E1197Q and E552Q ⁄ E1197Q were pre-incubated with5 lM 8-azido-[a32P]ATP, 3 mM MgCl2and 100 lM verapamil in theabsence (upper) or presence (lower) of 200 lM Vi for 20 min at37 °C. Unbound ligands were removed by ultracentrifugation andwashing, and the samples were then UV irradiated. The sampleswere promptly digested with trypsin (see Experimental proce-dures) at varying trypsin-to-protein ratios (lane 1, 1 : 75; lane 2,1 : 37.5; lane 3, 1 : 18.75; lane 4, 1 : 9.38; lane 5, 1 : 4.69; lane6, 1 : 2.34) and photolabeled, trypsinized samples were separatedby electrophoresis on 10% SDS polyacrylamide gels, transferredonto nitrocellulose membranes and subjected to autoradiography.The membranes were then analyzed by immunoblotting usingmouse anti-P-glycoprotein mAbs that recognize either the N-termi-nal half (MD13) or the C-terminal half (MD7), or both halves ofPgp (C219) (not shown) to identify fragments corresponding toNBD1 or NBD2, as indicated to the right.I. Carrier and P. Gros Abcb1a catalytic mechanismFEBS Journal 275 (2008) 3312–3324 ª 2008 The Authors Journal compilation ª 2008 FEBS 3317enzymes), we have created the following novelmutants: E552N, E1197N, E552Q ⁄ K1072R andK429R ⁄ E1197Q, to further characterize the role of theIC residues in the catalytic mechanism of Abcb1a. Asseen in Fig. 1, we were able to express and purify allmutants to high levels.Studies on the single-site mutantsOur results with the single-site mutants are reminiscentof those previously obtained with the glutamine muta-tion (E fi Q) [38,39]. Indeed, although all single-sitemutants show an absence of steady-state ATPase activ-ity, as measured by Pirelease, ATPase activity is notcompletely abolished and the mutants can cleave ATPto ADP and Piin a temperature-dependent fashion(Figs 3 and 4). The apparent lack of turnover is notdue to a major decrease in affinity by the enzymes forMgATP (Fig. 2). As suggested by Tombline et al. [38],very low turnover probably occurs in all the single-siteenzymes, but we have not used a more sensitive assayto determine that. Thus, as for the glutamine mutants,a step in the catalytic pathway must be substantiallyslowed, such that normal turnover is not observed bymeasuring Pirelease by our assay. The results obtainedwith the aspartate transformation (E fi D) areparticularly interesting because the 8-azidonucleotide-trapping properties of these enzymes with the mutationin NBD1 or NBD2 resemble the wild-type enzyme, butno steady-state ATPase activity was measured. Thus,the length of the IC residue side chain is important fornormal catalytic activity, but the presence of thecharge seems to slow a step further along the catalyticpathway, because the dependence on Vi for trapping isalmost normal. By contrast, when the charge isremoved, as in the glutamine (length of the side chainmaintained) and asparagine (shorter side chain)mutants, then trapping in the absence of Vi nowoccurs [39] (Fig. 3); this is also the case when the sidechain is almost completely removed as in the alaninemutants (Fig. 3). These results thus emphasize thestrict requirement for glutamate at this residue, withthe negative charge playing a crucial role. Anothernotable feature of the single-site IC mutants (includingthe glutamine substitutions) [39] is the fact that, in theabsence of Vi (± drugs) labeling of the enzymes withthe mutation in NBD2 is consistently lower than label-ing of the enzymes with the equivalent mutation inNBD1 (E fi A mutation in the presence of drug isan exception). However, the reverse is true when Vi ispresent in the labeling reaction; i.e. labeling of theenzymes with the mutation in NBD1 is consistentlylower than labeling of the enzymes with the equivalentmutation in NBD2. These observations hint at the factthat the two NBDs may not have the same affinity fornucleotide or that they may hydrolyze ATP at differentrates or in a given order. In all single-site mutants,drug stimulation can be observed, suggesting that sig-nal transduction between the drug binding site(s) andthe NBDs is not affected by the mutations.Studies on the double-mutantsIn this study, we also analyzed three double-mutants.First, we created the double-mutant in which the ICresidue is mutated to glutamine (E fi Q) in bothNBDs (Q ⁄ Q). Second, we created a mutant in whichNBD1 contains the E fi Q mutation and NBD2 con-tains the ATPase-inactivating mutation of the Walk-er A lysine (K1072R) (Q ⁄ R). Finally, the third double-mutant contains the E fi Q mutation in NBD2 andthe ATPase inactivating mutation of the Walker Alysine (K429R) is in NBD1 (R ⁄ Q). As seen in Fig. 3,these three double-mutants trap 8-azido-nucleotide in adrug-stimulated and Vi-independent fashion, but tovery different extents. The R ⁄ Q mutant enzyme is mostextensively labeled, followed by the Q ⁄ Q mutantenzyme and the Q⁄ R mutant enzyme, which showsalmost no labeling at all. Again, major changes inaffinity for 8-azido-ATP cannot account for the differ-ences in labeling with 8-azido-nucleotide (Fig. 2) andas in the single-site mutants, drug stimulation can beobserved (Fig. 3), suggesting that signal transductionbetween the drug-binding site(s) and the NBDs is notaffected by the mutations.When 8-azido-ADP production by the double-mutant enzymes is analyzed (Fig. 4), it is possible tosee that the R ⁄ Q and Q ⁄ R mutant enzymes do pro-duce ADP and this process is temperature sensitive,whereas the Q⁄ Q mutant enzyme does not produceany ADP. Based on previous results, it is tempting tosuggest that the Q ⁄ Q mutant enzyme is trapped in astable dimer in which nucleotide (ATP) is sandwichedat the interface. Our results with this mutant supportthis explanation. First, 8-azido-nucleotide labeling ofthis mutant does occur and appears to be completelyVi insensitive (Fig. 3). Second, this mutant appears notto produce ADP (Fig. 4). Finally, trapped 8-azido-nucleotide is observed in both NBDs (Fig. 6). TheR ⁄ Q and Q ⁄ R mutants do not appear to be trapped inthe same conformation as the Q ⁄ Q mutant. Deactiva-tion of the ‘wild-type’ NBD allows us to observe thatupon NBD dimerization only NBD2 can enter thetransition state. Thus, the results suggest that once thedimer is formed with nucleotide in each NBD, progres-sion into the transition state induces asymmetry in theAbcb1a catalytic mechanism I. Carrier and P. Gros3318 FEBS Journal 275 (2008) 3312–3324 ª 2008 The Authors Journal compilation ª 2008 FEBSdimer [47,48], such that NBD2 would be most likely tobe committed to hydrolyze its ATP. The conforma-tional change induced by hydrolysis at NBD2 wouldthen be transmitted to NBD1, which in turn would bein the correct conformation to hydrolyze its ATP,leading to full destabilization of the dimer. This sug-gests that the NBDs are not symmetrical and NBD2 isfirst committed to hydrolyze upon dimerization. Sucha scenario, in which hydrolysis is sequential in a closeddimer, does not invalidate the theory of alternate catal-ysis, but it must be taken into consideration that atransport cycle involves dimerization of the NBDs withhydrolysis of two nucleotides per dimerization andnot, as previously believed, a continuous turnovercomparable to a two-cylinder engine. Thus, the dimercloses with bound nucleotide in each active site, oneNBD is committed to hydrolysis (presumably NBD2)and hydrolyzes its nucleotide, then the other NBD(presumably NBD1) hydrolyzes its nucleotide andthese events cause conformational changes which leadto allocrite transport, destabilization of the NBDdimer and release of hydrolysis products, such that anew cycle can begin with the NBDs hydrolysing in thesame order, giving the impression of alternate sitecatalysis.Another very well-studied ABC transporter is the cys-tic fibrosis transmembrane conductance regulator(CFTR ⁄ ABCC7). Cystic fibrosis is a lethal disease thataffects about 1 in 2900 Caucasians and is caused bymutations in the CFTR ⁄ ABCC7 gene [49,50]. Althoughthe CFTR protein is part of the ABC superfamily ofproteins, it is not a classical ABC transporter, because itacts as a chloride channel. Despite or because of thispeculiarity, recent observations obtained by mutatingthe IC in CFTR’s NBD2 [51] seem to have unraveledsome of the mystery behind the catalytic cycle of ABCtransporters and also support our hypotheses. Thus, itappears that in CFTR, dimerization of the NBDsfollowing binding of ATP at both sites propagates asignal which leads to the opening of the chloridechannel [51]. Subsequent hydrolysis of ATP at the activenucleotide-binding site in NBD2 initiates channel clo-sure by destabilizing the NBD dimer. But, unlike typicalABC transporters, CFTR’s NBD1 is not ATPase activeand a possible explanation for the inactivation of thecatalytic activity with augmentation of affinity for ATPat NBD1 would be that this could maintain the NBDsin a closed dimer for longer, thus allowing the channelto be opened for a reasonable amount of time. The wayin which NBD1 may prolong channel opening couldeither be by delaying hydrolysis at NBD2 or becauseonce NBD2 has hydrolyzed, NBD1 still holds ATP andfull dimer dissociation is retarded. Transposing theseobservations to other ABC transporters, we can buildthe following hypothesis about catalytic activity: (a)ATP binds to both NBDs and forms a tight dimer, plau-sibly, this could be accelerated by drug binding to theTMDs; (b) as the dimer progresses towards the transi-tion state, conformational changes propagate to theTMDs and this allows the allocrite-binding site to ‘flip’the transport substrate from the high-affinity site to thelow-affinity site, (c) ATP hydrolysis is quickly initiatedat the NBDs and proceeds in a sequential fashion.Hydrolysis of ATP (one or both) may lead to furtherconformational changes required for full transport andthe release of allocrite. Presumably, ATP present atNBD1 induces ATP hydrolysis at NBD2 which is thenfollowed by hydrolysis at NBD1. (d) When only ADP ispresent, dimer destabilization occurs and NBDs moveapart, resetting the protein and releasing hydrolysisproducts (not Pias it can diffuse out freely onceformed).ConclusionsFrom the results obtained in this study, we would liketo suggest that once NBD dimerization has occurredwith one ATP molecule bound at each active site, pro-gression into the transition state induces asymmetry inthe nucleotide-binding sites such that NBD2 is com-mitted to hydrolysis.Analyzing the results of this and other studies, itseems that a dual role for the IC residues is starting toemerge; first the ICs appear to be important in NBD–NBD communication and transmission of the nucleo-tide state of one active site to the other; second, theICs appear to be involved in catalysis by contributingto the catalytic dyad along with the highly conservedH-loop His.Experimental proceduresAbcb1a cDNA modificationsAll mutations were created by site-directed mutagenesisusing a recombinant PCR approach as described previously[52]. Mutations in NBD1 at position E552 were introducedusing primer TK-5 (5¢-GTGCTCATAGTTGCCTACA-3¢)and the following mutagenic oligos: E552Ar (5¢-GTGGCCGCGTCCAAC-3¢), E552Dr (5¢-GTGGCGTCGTCCAAC-3¢)and E552Nr (5¢-AGGTGGCGTTGTCCAAC-3¢). A secondoverlapping mdr3 cDNA fragment was amplified usingprimer pairs HincII (5¢-GAAAGCTGTCAACGAAGCC-3¢) and primer Mdr3-2008r (5¢-CTGTGTCATGACAAGTTTG-3¢). The amplification products were purified on gel,mixed, denatured at 94 °C for 5 min followed by annealingat 54 ° C for 5 min and elongation at 72 °C for 5 minI. Carrier and P. Gros Abcb1a catalytic mechanismFEBS Journal 275 (2008) 3312–3324 ª 2008 The Authors Journal compilation ª 2008 FEBS 3319(repeated three times) with VENT DNA polymerase in areaction mixture without primers to generate hybrid DNAfragments. The hybrid products were then amplified usingprimers TK-5 and Mdr3-2008r and a 1113 bp MscI–SalIfragment carrying the mutated segment was purified andused to replace the corresponding fragment in the pVT–mdr3 construct [53] which had served as the template in thePCR. To screen for the desired mutations, individual plas-mids were isolated and the nucleotide sequence of the entire1113 bp MscI–SalI fragment was determined. The muta-tions were then transferred to pHIL–mdr3.5–His6[24] usingthe restriction enzymes AflII and EcoRI, as previouslydescribed [35]. Mutations in NBD2 at position E1197 wereintroduced using primer Y1040Wf (5¢-GTGTTCAACTGGCCCACCCG-3¢) and the following mutagenic oligos:E1197Ar (5¢-GATGTTGCTGCGTCCAGAAG-3¢), E1197Dr (5¢-GATGTTGCATCGTCCAGAAG-3¢) and E1197Nr(5¢-GATGTTGCGTTGTCCAGAAG-3¢). A second over-lapping mdr3 cDNA fragment was amplified using muta-genic oligos E1197Af (5¢-CTGGACGCAGCAACATC-3¢),E1197Df (5¢-CTGGACGATGCAACATC-3¢) and E1197Nf(5¢-CTGGACAACGCAACATCAG-3¢) with primer pHIL–3¢r(5¢-GCAAATGGCATTCTGACATCC-3¢). The amplifi-cation products were purified on gel, mixed, denatured at94 °C for 5 min followed by annealing at 52 °C for 5 minand elongation at 68 °C for 5 min (repeated three times)with Taq HiFi polymerase in a reaction mixture withoutprimers to generate hybrid DNA fragments. The hybridproducts were then amplified using primers Y1040Wf andpHIL–3¢r and a 617 bp XhoI(3386)–XhoI(4003) fragmentcarrying the mutated segment was purified and used toreplace the corresponding fragment in the pHIL–mdr3.5–His6construct which had served as template in the PCR.To screen for the desired mutations and correct orientationof the inserted fragment, individual plasmids were isolatedand the nucleotide sequence of the entire 617 bpXhoI(3386)–XhoI(4003) fragment was determined. For thedouble-mutant E552Q ⁄ E1197Q, the E552Q mutation wasexcised from pHIL–E552Q using the restriction enzymesXmaI and EcoRI and the 485 bp fragment containing themutation was introduced in the corresponding sites ofpHIL–E1197Q. For the double-mutant E552Q ⁄ K1072R, theK1072R mutation was introduced into the E552Q templateusing a standard PCR approach with primer HincII andthe mutagenic oligo which contains the XhoI site K1072R–XhoIr (5¢-CCGCTCGAGCAGCTGGACCACTGTGCTCCTCCCGC-3¢). The 1622 bp XmaI–XhoI fragment contain-ing both mutations was then introduced into pHIL–Mdr3.To screen for the desired mutations, individual plasmidswere isolated and the nucleotide sequence of the entire1622 bp XmaI–XhoI fragment was determined. For the dou-ble-mutant K429R ⁄ E1197Q, a recombinant PCR techniquewas used to create the K429R mutation using pHIL–mdr3.5as template. A first fragment was created using primerMdr3-1202f (5¢-TTCGCCAATGCACGAGG-3¢) and muta-genic oligo K429Rr (5¢-GTTGTGCTTCTTCCACAG-3¢).A second overlapping mdr3 cDNA fragment was amplifiedusing mutagenic oligo K429Rf (5¢-CTGTGGAAGAAGCACAAC-3¢) and primer E552Qr (5¢-GGTGGCCTGGTCCAACAAAAG-3¢). The amplification products were purifiedon gel, mixed, denatured at 98 °C for 5 min and allowedto cool slowly to room temperature in a reaction mixturewithout primers to generate hybrid DNA fragments.Klenow polymerase and dNTPs were added to fill-in thesingle-stranded overhangs. The hybrid products were thenamplified with VENT DNA polymerase using primersMdr3-1202f and E552Qr and a 402 bp BglII–XmaI fragmentcarrying the mutated segment was purified and used toreplace the corresponding fragment in the pHIL–E1197Qconstruct. To screen for the desired mutation, individualplasmids were isolated and the nucleotide sequence of theentire 402 bp BglII–XmaI fragment was determined.Purification of Abcb1aFor expression and purification of the six single and threedouble mutants, pHIL–mdr3–His6or pHIL–mdr3.5–His6carrying either a wild-type or mutant version of Abcb1awas transformed into P. pastoris strain GS115, accordingto the manufacturer’s instructions (Invitrogen, Carlsbad,CA, USA; license number 145457) and screened for expres-sion as previously described [35]. Glycerol stocks of P. pas-toris GS115 transformants were streaked on YPD platesand single colonies were used to inoculate 6 L liquid cul-tures. For preparation of P. pastoris membranes, cultureswere induced with 1% methanol for 72 h and plasma mem-branes were isolated by centrifugation, as described previ-ously [41]. Solubilization and purification of wild-type andmutant Abcb1a variants by affinity chromatography onNi-NTA resin (Qiagen, Valencia, CA, USA) and DE52-cel-lulose (Whatman, Florian Park, NJ, USA) was as describedpreviously [41]. This procedure routinely yielded between0.4 and 2.5 mg of protein, with 95% minimum purity.Assay of ATPase activityFor ATPase assays, purified wild-type or mutant Abcb1aenzymes (concentrated DE52 eluate) were activated by incu-bating with 0.5% E. coli lipids (w ⁄ v; Avanti, Alabaster, AL,USA acetone ⁄ ether preparation; equivalent to 50 : 1 w ⁄ wlipid to protein ratio) and 5 mm dithiothreitol for 30 min at20 °C at a final protein concentration of 0.07 lgÆlL)1(wild-type) or 0.1 lgÆlL)1(mutants). Aliquots of 5 lL were addedinto 50 mm Tris ⁄ HCl (pH 8.0), 0.1 mm EGTA, 10 mmNa2ATP and 10 mm MgCl2, to a final volume of 250 lLand the mixture was incubated at 37 °C. At the appropriatetime, a 50 lL aliquot was removed and quenched in 1 mLof ice-cold 20 mm H2SO4. Inorganic phosphate (Pi) releasewas assayed as described previously [42]. Drugs were addedAbcb1a catalytic mechanism I. Carrier and P. Gros3320 FEBS Journal 275 (2008) 3312–3324 ª 2008 The Authors Journal compilation ª 2008 FEBSas dimethylsulfoxide stock solutions and the final solventconcentration in the assay was kept at £ 2% (v ⁄ v).Photoaffinity labeling with 8-azido-[a32P]ATP8-Azido-[a32P]ATP photoaffinity labeling was performed asdescribed previously [35] with minor modifications. The puri-fied Abcb1a proteins (concentrated DE52 eluate) were acti-vated by incubating with E. coli lipids at a 50 : 1 lipid ⁄protein ratio (w⁄ w; Avanti, acetone ⁄ ether preparation) and5mm dithiothreitol, at a final concentration of 0.2 mgÆmL)1,at 20 °C for 30 min immediately prior to starting the phot-olabeling reactions. For direct labeling experiments, acti-vated wild-type or mutant Abcb1a variants were incubatedon ice for  10 min with 3 mm MgCl2,50mm Tris ⁄ HCl(pH 8.0), 0.1 mm EGTA and varying concentrations of8-azido-[a32P]ATP (5, 20 and 80 lm final concentrations at0.2 CiÆmmol)1specific activity) in a total volume of 50 lL(3 lg protein per sample). The samples were kept on ice andimmediately UV-irradiated for 5 min (UVS-II Minerallight,260 nm, placed directly above the samples). Unreacted nucle-otides were then removed by centrifugation at 200 000 g for30 min at 4 °C in a TL-100 rotor (Beckman, Mississauga,Canada) and protein-containing pellets were washed with100 lL ice-cold 50 mm Tris ⁄ HCl (pH 8.0) and 0.1 mmEGTA. The pellets were dissolved in sample buffer (5% w ⁄ vSDS, 25% v ⁄ v glycerol, 0.125 m Tris ⁄ HCl pH 6.8, 40 mmdithiothreitol, 0.01% pyronin Y) and separated by SDS ⁄PAGE on 7.5% gels, followed by autoradiography to KodakBioMax MS film (Eastman Kodak Co., Rochester, NY,USA). For nucleotide-trapping experiments, activated wild-type or mutant Abcb1a variants were incubated at 37 °C for20 min with 5 lm 8-azido-[a32P]ATP, 3 mm MgCl2,50mmTris ⁄ HCl (pH 8.0) and 0.1 mm EGTA, with or without vana-date (Vi, 200 lm) in a total volume of 50 lL(3lg proteinper sample). Verapamil (100 lm) or valinomycin (100 lm)were included where indicated. Modifications to the normalprocedure are indicated in the figure legends. The incubationswere started by addition of 8-azido-[a32P]ATP and stoppedby transfer on ice. Free label was then removed by centrifu-gation at 200 000 g for 30 min at 4 °C in a TL-100 rotor(Beckman) and pellets were washed and resuspended in30 lL of ice-cold 50 mm Tris ⁄ HCl (pH 8.0) and 0.1 mmEGTA. Samples were kept on ice and irradiated with UVlight for 5 min. Labeled samples were resolved by SDS ⁄PAGE on 7.5% gels and subjected to autoradiography.Orthovanadate solutions (100 mm) were prepared from Na3VO4(Fisher Scientific, Pittsburgh, PA, USA) at pH 10 andboiled for 2 min before use to break down polymeric species.TLC analysis of vanadate-trapped nucleotidesin Abcb1aTLC was performed exactly as described in Carrier et al.[39].Partial trypsin digestion of photolabeled Abcb1aIn order to detect radiolabeled nucleotide trapped in NBD1and ⁄ or NBD2 of Abcb1a following photolabeling of theprotein with 8-azido-[a32P]ATP in the presence or absenceof Vi, we took advantage of the protease hypersensitive sitelocated in the linker region joining the two halves of Pgp[54]. Photoaffinity-labeled proteins were resuspended in30 lLof50mm Tris ⁄ HCl (pH 8.0) and 0.1 mm EGTA andkept on ice. The incubation with trypsin (2 lL of eachstock solution) was carried out for 6 min at 37 °Catenzyme-to-protein mass ratios of 1 : 75, 1 : 37.5, 1 : 18.75,1 : 9.38, 1 : 4.69 and 1 : 2.34. Digestion was stopped by theaddition of 15 lL of sample buffer. Finally, the Abcb1afragments were resolved by SDS ⁄ PAGE on 10% gels, fol-lowed by transfer to nitrocellulose membranes and exposi-tion to film. Immunoblotting with the mouse mAb C219(Signet Laboratories Inc., Dedham, MA, USA) that reco-gnizes both halves of Abcb1a, as well as with N- andC-terminal half specific mouse mAbs [MD13 with itsepitope in NBD1 (494–504) and MD7 with its epitope inthe intracellular loop 3 (805–815)], respectively (gift ofV. Ling, The B.C. Cancer Research Centre, Vancouver,Canada) [55] was then performed on the membranes.Routine proceduresProtein concentrations were determined by the bicinchoni-nic acid method in the presence of 0.5% SDS using BSA asa standard. SDS ⁄ PAGE was carried out according toLaemmli [56] using the mini-PROTEAN II gel and Electro-transfer system (Bio-Rad Labs, Hercules, CA, USA).Samples were dissolved in sample buffer (5% SDS w ⁄ v,25% glycerol v ⁄ v, 125 mm Tris ⁄ HCl pH 6.8, 40 mm dithio-threitol and 0.01% pyronin Y). For immunodetection ofAbcb1a, the mouse mAb C219 (Signet) was used withthe enhanced chemiluminescence detection system (NENRenaissance, Perkin–Elmer, Wellesley, MA, USA). To rec-ognize NBD1 specifically, the mouse mAb MD13 was usedand for NBD2 the mouse mAb MD7 was employed. Forautoradiography, SDS gels were stained with CoomassieBrilliant Blue, dried and exposed at )80 °C to KodakBioMax MS film with an intensifying screen for theappropriate time.Materials8-Azido-[a32P]ATP was purchased from Affinity LabelingTechnologies, Inc. (Lexington, KY, USA). 8-Azido-ATPand verapamil were from ICN (Costa Mesa, CA, USA),and valinomycin was from Calbiochem (San Diego, CA,USA). Acetone ⁄ ether-precipitated E. coli lipids were fromAvanti Polar Lipids. The PEI-cellulose TLC plates and gen-eral reagent grade chemicals were from Sigma (St. Louis,MO, USA) or Fisher Scientific (Pittsburgh, PA, USA).I. Carrier and P. Gros Abcb1a catalytic mechanismFEBS Journal 275 (2008) 3312–3324 ª 2008 The Authors Journal compilation ª 2008 FEBS 3321[...]... 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(1995) P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site J Biol Chem 270, 19383–19390 Tombline G, Muharemagic A, White LB & Senior AE (2005) Involvement of the ‘occluded nucleotide conformation’ of P-glycoprotein in the catalytic pathway Biochemistry 44, 12879–12886 Ivetac A, Campbell JD & Sansom MS (2007) Dynamics and function in a bacterial ABC... contribution to the DNA binding activity of the Pax-3 paired domain J Biol Chem 272, 28289–28295 53 Beaudet L & Gros P (1995) Functional dissection of P-glycoprotein nucleotide-binding domains in chimeric 3324 and mutant proteins Modulation of drug resistance profiles J Biol Chem 270, 17159–17170 54 Bruggemann EP, Germann UA, Gottesman MM & Pastan I (1989) Two different regions of P-glycoprotein [corrected].. .Abcb1a catalytic mechanism I Carrier and P Gros Acknowledgements We are grateful to Dr Victor Ling (The B.C Cancer Research Centre, Vancouver, Canada) for the generous gift of the mouse mAbs MD13 and MD7 This study ´ was supported by an FRSQ-FCAR-Sante scholarship to IC and by research grants to PG from the Canadian Institute of Health Research (CIHR) PG is a Career Scientist of the CIHR of Canada . Investigating the role of the invariant carboxylate residues E552 and E1197 in the catalytic activity of Abcb1a (mouse Mdr3) Isabelle Carrier and Philippe. investigatedfurther the role of these two IC residues in the catalytic mechanism of Abcb1a. For this, wild-type and the Abcb1a mutants E552D, E552N, E552A,E1197D,
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